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Brassica oleraceae
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italica
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Lukas Mulyono Samuel*, Francisca AS**, Khie Khiong***
*Division of Gastroentero-hepatology, Department of Internal Medicine, Faculty of Medicine, University of Christian Maranatha/Immanuel Hospital, Bandung
**Faculty of Medicine, University of Christian Maranatha, Bandung ***Department of Biology, Faculty of Medicine, University of Christian
Maranatha, Bandung
Corresponding author:
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Background:8OFHUDWLYHFROLWLV8&LVDERZHOLQÀDPPDWLRQWKDWKDSSHQVLQWKHFRORQDQGUHFWXP,Q 8&WKHUHLVDQLQFUHDVHGOHYHORILQÀDPPDWRU\PHGLDWRUVRQHRIZKLFKLVLQWHUOHXNLQ,/WKHPDLQ FKHPRDWWUDFWDQWIRUSRO\PRUSKRQXFOHDU301LQÀDPPDWRU\FHOOVFDXVLQJLQÀDPPDWLRQWRZRUVHQ%URFFROL %UDVVLFDROHUDFHDH/YDULWDOLFDFRQWDLQVVXOIRUDSKDQHZKLFKFDQLQKLELWQXFOHDUIDFWRUNDSSD%1)Ƹ% resulting in the decrease of IL-8. This study was performed to identify the effect of steamed broccoli juice to the serum interleukin-8 level in murine model of colitis.
Method:7KLVVWXG\ZDVDWUXHH[SHULPHQWDOODERUDWRU\VWXG\XVLQJFRPSOHWHUDQGRPL]HGGHVLJQ([SHULPHQWDO animals being used were 28 murines with Balb/c strain and were divided into 4 intervention groups. Serum IL-8 level was measured by using enzyme-linked immunosorbent assay (ELISA) method. Data analysis was performed XVLQJDQDO\VLVRIYDULDQFH$129$WHVWIROORZHGE\PXOWLSOHFRPSDULVRQV7XNH\KRQHVWVLJQL¿FDQWGLIIHUHQFH (HSD) test (D DQGS<
Results: The highest mean of IL-8 level was found in the positive control group. This study also showed highly VLJQL¿FDQWGLIIHUHQFHSRI,/OHYHOEHWZHHQSRVLWLYHFRQWUROJURXSDQGWUHDWPHQWJURXS
Conclusion:Steamed broccoli juice could decrease serum interleukin-8 level in murine model of colitis.
Keywords: ulcerative colitis, sulforaphane, interleukin-8
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Latar belakang: Kolitis ulseratif (KU) adalah peradangan usus yang terjadi pada kolon dan rektum. Pada .8WHUMDGLSHQLQJNDWDQNDGDUPHGLDWRULQÀDPDVLVDODKVDWXQ\DDGDODKLQWHUOHXNLQ,/\DQJPHUXSDNDQ kemoatraktan utama untuk sel radang polymorphonuclear (PMN), menyebabkan peradangan semakin parah. Brokoli (Brassica oleraceae L. var. italica) mengandung sulphoraphane yang dapat menghambat nuclear factor NDSSD%1)Ƹ%VHKLQJJDWHUMDGLSHQXUXQDQ,/3HQHOLWLDQLQLGLODNXNDQXQWXNPHQJHWDKXLSHQJDUXKVDUL kukusan brokoli terhadap kadar interleukin-8 serum pada mencit model kolitis.
(ELISA). Analisis data dilakukan dengan menggunakan analisis varian (ANOVA) satu arah, dilanjutkan dengan uji multiple comparisons 7XNH\KRQHVWVLJQL¿FDQWGLIIHUHQFH+6'D GDQS<
Hasil:Rerata kadar IL-8 yang paling tinggi terdapat pada kelompok kontrol positif. Penelitian ini juga PHQXQMXNNDQSHUEHGDDQ\DQJVDQJDWVLJQL¿NDQSNDGDU,/DQWDUDNHORPSRNNRQWUROSRVLWLIGDQ kelompok terapi.
Simpulan: Sari kukusan brokoli dapat menurunkan kadar interleukin-8 serum pada mencit model kolitis.
Kata kunci: kolitis ulserativa, sulforaphane, interleukin-8
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,QÀDPPDWRU\ERZHOGLVHDVH(IBD) is an idiopathic
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of Chron’s disease (CD) and ulcerative colitis (UC). Pathogenesis of these two diseases has not been fully understood. Genetic and environmental factors hold important role in the disregulation of intestinal immunity which further causes trauma to the digestive
tract.1 UC is the most frequent form found in IBD
patients. The incidence of UC worldwide reaches 1.2-20.3 cases per 100,000 individual per year. The highest incidence and prevalence of IBD were found in America and North Europe, while the lowest incidence and prevalence were found in Asia. However, based on the available data, there was increased incidence of IBD in Asia. This is associated with the environmental condition and life style in Western Countries, which may cause the incidence of IBD, such as: smoking, high fat diet, sugar, stress, particular drugs, and the
high socio-economic status.2,3 Data from endoscopy
unit in several hospitals in Jakarta revealed IBD cases with chronic diarrhea 12.2%, IBD with haematochezia 3.9%, IBD with stomachache 2.8%, and IBD with chronic diarrhea, haematochezia, and stomachache
25.9%.4
Pathogenesis of UC is often associated with the
SUHVHQFH RI LQFUHDVHG LQÀDPPDWRU\ PHGLDWRUV DQG
oxidative stress.5 Nuclear factor kappa B (NF-ËB) is a F\WRNLQHUHJXODWRUDQGSURLQÀDPPDWRU\FKHPRNLQH
One of the main chemokines is interleukin 8 (IL-8).6
The level of IL-8 in the intestinal mucosa of patients with UC is higher compared to patients with CD. The increase of IL-8 is in accordance with the severity
of disease histopathologically.7,8 IL-8 is the main
chemoattractant in CD and UC.9,10,11
Broccoli (Brassica oleracea L. var. Italica)
belongs to the family Brassicacea. Broccoli contains sulforaphane (SFN) substance which has protection
benefit towards bacteria, cancer, and diabetes.12
Previous studies showed that administration of broccoli to murine might give preventive effect towards
colitis induction by dextran sulphate sodium (DSS) in the form of amelioration in diarrheal degree and
clinical score.13 SFN which is contained in broccoli
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cytokine produced by macrophage in bacteria infected
murine model.14 SFNhas been studied to have
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further cause the release of phase II enzyme and is
SURGXFHGE\QRQWR[LFDJHQWWRLQKLELWLQÀDPPDWRU\ SURFHVV$QWLLQÀDPPDWRU\PHFKDQLVPRI6)1LVDOVR DVVRFLDWHG ZLWK WKH LQKLELWLRQ RI SURLQÀDPPDWRU\ PROHFXOHVLJQDOIURP1)Ƹ%,QYLWURVWXG\VKRZHG
that SFN decreased the release of interleukin, and TNF-DWKURXJKGLUHFWLQKLELWLRQRI1)Ƹ%SDWKZD\
ZKLFKIXQFWLRQLQUHJXODWLRQRILQÀDPPDWRU\F\WRNLQHV DQG LQÀDPPDWRU\ FKHPRNLQHV13,15,16 Until recently,
there is no study of steamed broccoli juice as a treatment of UC. Based on the aforementioned things, this study was performed to know the role of steamed broccoli juice in decreasing serum IL-8 level in colitis murine model. The novelty of this study was to identify how steamed broccoli juice colitis could play role as a curative agent in colitis murine model by suppressing IL-8 pathway as a chemotaxis pathway.
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This study was a true experimental laboratory study with complete randomized design. The serum IL-8 level was measured by using enzyme-linked immunosorbent assay (ELISA) and was read using ELISA plate reader with 450 nm wavelength. Experimental animals being used were 28 male murines BALB/c strain aged 8 weeks, with weight ranged around 25 grams, divided into 4 groups of intervention (n = 6) based on the formula (t-1) (n-1)
17 in which t is the total intervention group and n
colitis group consisted of positive control and broccoli treatment groups were given DSS 2.5% (w/v) as much as 0.5 mL through oral route using nasogastric tube. On day-15 all murines’ serum blood were taken for serum IL-8 level examination. Data was obtained in pg/mL, analysed using one way analysis of variance (ANOVA) test continued with multiple comparisons
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D = 0.05 using SPSS version 17 software, in which a
GLIIHUHQFHZHUHPDUNHGWREHVLJQL¿FDQWLISd 0.05.
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Serological examination by using ELISA method to all serum murine groups which had been given steamed broccoli juice intervention for 7 days showed the mean of IL-8 level as shown in Table 1.
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1& %& 3& %
1 0.184 3.870 16.339 7.848 2 0.000 1.435 15.996 6.528 3 0.000 3.576 12.765 7.848 4 0.259 1.078 18.335 6.142 5 0.430 0.110 17.971 7.220 6 0.259 0.120 18.551 11.653 Mean 0.188 1.698 16.660 7.873 STDEV 0.167 1.655 2.182 1.975
NC: negative control (administration of aquadest ad libitum); BC: broccoli control (administration of steamed broccoli juice 1.5 mL); PC: positive control (administration of DSS 2.5% (w/v)); B: broccoli treatment (administration of DSS 2.5% (w/v) followed by steamed broccoli juice 1.5 mL)
Mean IL-8 data (Table 1) in every treatment showed that lowest serum IL-8 level was found in negative control (NC) group, while highest IL-8 level was found in positive control group, which was induced by DSS without administration of steamed broccoli juice.
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VTXDUHV GI VTXDUH0HDQ ) p
Between groups
1007.707 3 335.902 117.483 0.000 Within
groups
57.183 20 2.859 Total 1064.890 23
F count = 117.483 > F table 0.05 (3.20) = 3.10 with p = 0.00
ĮWKHUHIRUH+0 was rejected, and H1 was accepted, meaning that there was difference of serum IL-8 level between at least 1 pair of intervention group. Further, to determine which of it between different groups, Post Hoct test was performed using Tukey HSD method. Results of Tukey HSD analysis could be seen in Table 3 and Figure 1.
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7UHDWPHQW
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NC 0.430ns 0.000** 0.000**
BC 0.000** 0.000**
PC 0.000**
B
NC: negative control (administration of aquades ad libitum); BC: broccoli control (administration of steamed broccoli juice 1.5 mL); PC: positive control (administration of DSS 2.5% (w/v)); B: broccoli treatment (administration of DSS 2.5% (w/v) followed with steamed broccoli juice 1.5 mL); ns: not VLJQL¿FDQWKLJKO\VLJQL¿FDQW
Based on the results of Tukey HSD test (Table
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difference of IL-8 level (p < 0.01) between negative control (NC) and positive control (PC) and broccoli treatment (B) groups. Further, there was also not
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= 0.00) in DSS group compared to the broccoli control (BC) and broccoli treatment (B) groups with p value < 0.01 in each groups.
Comparison of each experimental group can be seen in Figure 1 below.
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NC: negative control (administration of aquades ad libitum); BC: broccoli control, administration of steamed broccoli juice 1.5 mL; PC: positive control (administration of DSS 2.5% (w/v)); B: broccoli treatment (administration of DSS 2.5% (w/v) followed with steamed broccoli juice 1.5 mL); NS: not VLJQL¿FDQW+LJKO\6LJQL¿FDQW
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Results of this study showed that DSS might induce the occurrence of colitis in murine. This was supported with the presence of increased IL-8 level. Table 1 revealed the mean of IL-8 in positive control group, which was given DSS 2.5% markedly increased compared to those with negative control group. Table 2 also showed the presence of difference of IL-8 level
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there was also increased IL-8 serum level. This was caused by the murines being used were not sterile murine and the room in which the murines’ cages were put were also not sterile, thus there could be a possibility of exposures to microbes or other substances which could cause increased of IL-8.
This study also revealed that steamed broccoli
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of steamed broccoli juice to colitis murine model can decrease the level of IL-8. In Table 1, it was shown that the lowest mean of IL-8 level was found in negative control group, while the highest was found in positive control groups. However, in groups receiving intervention, IL-8 level was found to be lower compared to the positive control groups. Table
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level (p = 0.000) between negative control groups and positive control group and broccoli treatment group. Additionally, in Table 3, it was shown that
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(p = 0.000) between positive control and broccoli treatment groups. This showed that steamed broccoli juice could lower IL-8 level. Difference of IL-8 level in each groups was also shown in Figure 1.
UC is marked by the mucosal damage and ulceration involving the rectum and spreading to the colon. In UC, there was involvement of mediators,
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Interleukin-6. These mediators cause the occurrence of clinical inflammatory symptoms, particularly
diarrhoea and weight loss.16 Other mediators which
cause inflammation are chemokines. One of the chemokines playing important role was IL-8. IL-8 was
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particularly neutrophils. The presence of neutrophil
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to be more severe which is shown in clinical or even
histopathological appearance.11
Broccoli contains sulforaphane that is a derivate of isothiocyanate and has chemopreventive effect towards
LQÀDPPDWLRQDQGFDQFHU2QHRIWKHPHFKDQLVPVRI VXOIRUDSKDQHLVE\LQKLELWLQJ1)Ƹ%ZKLFKLVWKHPDLQ UHJXODWRURILQÀDPPDWRU\F\WRNLQHDQGFKHPRNLQH 6)1VHOHFWLYHO\LQKLELW'1$ELQGLQJWR1)Ƹ%HLWKHU
direct or indirectly. Directly, SFN inhibit degradation
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indirectly through the interaction with redox regulator including thioredoxin and Ref-1 which was associated
ZLWK1)ɤ%DFWLYLW\18,19 SFN which is associated with
Nrf-2 will activate phase II enzymeswhich further can
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Based on the explanation above, the administration of steamed broccoli juice for 7 days might decrease the IL-8 serum level in colitis murine models which were induced by DSS. This study explained how was the curative effect of broccoli towards IL-8 in colitis murine model. Application of this research in the future was that broccoli could be used as a complementary treatment in patients with ulcerative colitis. Researches on the effect of broccoli towards clinical score and
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as TNF-a are needed to further explain the molecular mechanism underlying the inhibitory process of
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Steamed broccoli juice decreased serum IL-8 serum level in colitis murine model.
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1. %HUQVWDLQ &1 )ULHG 0 &RKHQ + ,QÀDPPDWRU\ %RZHO
Disease: A Global Perspective. World Gastroenterology Organisation Global Guidelines, 2009.p.112-24.
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DFWLYLW\RIOLYHUGHWR[L¿FDWLRQ-XUQDO.HGRNWHUDQ0DUDQDWKD
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